This invention relates to a Fas ligand, a part of the Fas ligand and a novel DNA fragment which encodes the Fas ligand, which may be utilized in the field of medicines. This invention also relates to an antibody which can be used for detecting of the Fas ligand, which may be utilized in the field of diagnostic reagents. This invention also relates to a recombinant DNA molecule which contains the novel DNA fragment, a transformant, a process for purifying the novel protein and a process for producing of the novel protein. This invention also relates to an antisense oligonucleotide.
Based on the morphology of dying cells, Cell death is generally divided into two categories, necrosis and apoptosis. In the necrotic cell death, degradation of cell membrane and release of cellular contents into extracellular matrix are observed. On the other hand, in the apoptotic cell death, fragmentation of chromosomal DNA and concentration of nuclei are observed while the degradation of the cell membrane and the release of the cellular contents as observed in the necrosis are not observed. Apoptosis has been conceived to be a form of programmed cell death. For example, phenomena of deletion of unnecessary cells and organs in the course of ontogenesis are considered to be caused by the apoptosis of cells. Another phenomenon that is considered to be the apoptosis is the cell death which occurs when virus-infected cells and tumor cells are attacked and removed by cytotoxic T cells (CTL), natural killer cells (NK cells), TNF-xcex1, TNF-xcex2 and the like. As described above, apoptosis is one of the physiological phenomena which have recently attracted special attention, and studies are being conducted by many researchers with the aim of elucidating the function of the apoptosis as well as its physiological meaning and relation to diseases.
Fas antibody has been known as a substance which can induce the apoptosis of cells. Fas antibody is a monoclonal antibody obtained by immunizing a mouse with human fibloblast (Yonehara S. et al., J. Exp. Med., vol. 169, pp. 1747-1756, 1989). Since Fas antibody has been obtained by the immunization of a mouse with cells, the type of molecules recognized by Fas antibody and the transfer mechanism of apoptosis signal to the cells were not revealed for a long time. Recently, however, Itoh N. et al. have succeeded in cloning the gene of the molecule (Fas antigen) which is specifically-recognized by the Fas antibody (Cell, vol. 66, pp. 233-243, 1991). It was then found that the Fas antigen is a cell membrane protein having a size of about 45 kD, and its amino acid sequence analysis has revealed that the Fas antigen belongs to the family of TNF receptors. In addition, a mouse Fas antigen gene was cloned (Watanabe-Fukunaga R. et al., J. Immunol., vol. 148, pp. 1274-1279, 1992), and the mRNA for Fas antigen was confirmed to be expressed in thymus, liver, lung, heart and ovary of the mouse.
After the cloning of the Fas antigen gene, a number of studies have been conducted and reported on the relationship between the apoptosis mediated by Fas antigen and various diseases.
Kobayashi N. et al. have reported that expression of Fas antigen on T cell membrane is induced upon infection with AIDS virus, suggesting a possibility that the apoptosis of T cells found in AIDS is a Fas antigen-mediated phenomenon (Nikkei Science, vol. 6, pp. 34-41, 1993).
Ogasawara J. et al. have observed that a phenomenon similar to fulminant hepatitis occurs upon administration of Fas antibody to a mouse, and suggested a possibility of the occurrence of a Fas antigen-mediated apoptosis in the inflammatory lesion of the fulminant hepatitis or the like (Nature, vol. 364, pp. 806-809, 1993). Hiramatsu N. et al. have reported that, in a patient suffering chronic type C hepatitis, Fas antigen is frequently expressed in the inflammatory lesion of the liver where leukocyte infiltration is observed (Hepatology, vol. 19, pp. 1354-1359, 1994).
In addition, Watanabe-Fukunaga R. et al. have confirmed that a mutation in the Fas antigen gene is present in the lpr mouse, which is one of autoimmune disease model animals and that the cells expressing such mutation in the Fas antigen gene do not undergo the apoptosis (Nature, vol. 356, pp. 314-317, 1992). Watanabe-Fukunaga R. et al. have estimated that autoimmune disease-like symptoms are generated by the autoreactive T cells remaining in the body, that should have been removed from the body through the apoptosis.
As described above, a number of studies have been reported on the relation between the Fas antigen and diseases. An unsolved question is whether or not there is a molecule (Fas ligand) that binds to the Fas antigen on the cell surface to induce the cell apoptosis, namely, a molecule that acts in a manner similar to the above described Fas antibody.
Watanabe-Fukunaga R. et al. have presumed that, in a gld mouse which exhibits autoimmune disease-like symptoms, an abnormality should be present in a biological molecule which binds to Fas antigen, as in the case of the lpr mouse.
In addition, Rouvier E. et al. have reported that a certain type of T cells show a specific cytotoxic action on the cells expressing Fas antigen (J. Exp. Med., vol. 177, pp. 195-200, 1993). More illustrating, they have shown that a Ca2+-independent cytotoxic action by mouse peripheral blood lymphocytes (PBL) and PC60-d10S cells, the latter being a hybridoma of mouse CTL and rat T-lymphoma cells, is observed only for the Fas antigen-expressing cells. They have also suggested the possibility that such lymphocytes, especially T cells, could recognize the Fas antigen or certain Fas antigen-related molecules, and the possibility that these cells might express to Fas ligand.
Though the possibility of the presence of a Fas ligand has been suggested by the researchers as described above, its specific nature is not yet revealed.
It is important to isolate the Fas ligand in order to elucidate mechanism of the Fas antigen-mediated apoptosis and reveal relationship between the Fas antigen-mediated apoptosis and diseases more clearly. In consequence, confirmation of the presence of the Fas ligand and revelation of its true nature have been called for in various fields including the medicine.
A primary object of the present invention is to provide the field of medical care with a Fas ligand and its gene and a means for artificially regulating apoptosis generated in the body.
As described in the foregoing, the diseases considered to be related to the Fas antigen-mediated apoptosis include those which are caused by the Fas antigen-mediated apoptosis, and, those which are caused by the absence of the Fas antigen-mediated apoptosis. For example, it is estimated that death of hepatocytes and the subsequent reduction in the liver function in the case of hepatitis and decrease in the number of HIV-infected T cells and the subsequent reduction in the immunological function in the case of AIDS would be improved by inhibiting the apoptosis. In the case of certain autoimmune diseases, on the other hand, the symptoms are estimated to be improved by allowing the Fas antigen-mediated apoptosis to occur normally and by enhancing the removal of autoantigen reactive T cells. With regard to the treatment of AIDS in its early stage, induction of the apoptosis of the cells infected with HIV and their removal from the body would be effective. Morimoto H. et al. have reported that the Fas antigen-mediated apoptosis induced in cancer cells could synergistically enhance carcinostatic effects of adriamycin and cisplatin (Cancer Res., vol. 53, pp. 2591-2596, 1993). A substance capable of binding to Fas antigen to induce apoptosis should be useful in the treatment of cancers.
Such a treatment which is based on the principle of artificially regulating the Fas antigen-mediated apoptosis can be established only after the identification of the Fas ligand. In other words, artificial enhancement of the apoptosis in the living body would be enabled when the Fas ligand is specified.
In order to use the Fas ligand or a part of the ligand in medical treatments and researches, it would be necessary to produce the Fas ligand protein in a large scale and at a high purity. Cloning of the gene coding for such protein will enable the production of the protein by means of genetic engineering techniques, and the thus produced protein can be used for the main effective component in therapeutic drugs and in the production of antibodies. In addition, the gene itself could be used in gene therapy and development of antisense drugs, as well as for the preparation of model animals, such as transgenic mice, of apoptosis-related diseases.
The inventors of the present invention have conducted intensive studies with the aim of isolating -Fas ligand, taking note of the aforementioned PC60-d10S cells. PC60-d10S is a hybridoma of mouse CTL and rat T-lymphoma, which induces apoptosis only in Fas antigen-expressing cells when stimulated with PMA (phorbol myristate acetate) and ionomycin. As a result of intensive studies, the present inventors have succeeded in obtaining a cell population, PC60-d10S-2, which shows higher cytotoxicity without stimulation and is useful for the cloning of the Fas ligand gene. Thereafter, the rat Fas ligand gene was cloned making use of this cell population. Also, th e present inventors have conducted intensive studies with the aim of isolating a human Fas ligand suitable for use in pharmaceutical drugs and the like and succeeded in obtaining a gene which encodes human Fas ligand, as well as a gene which encodes mouse Fas ligand. It was confirmed thereafter that these genes have similar sequences having a common partial sequence. The inventors of the present invention have also identified the part of the sequence that should be necessary in exhibiting Fas ligand function. The present invention has been accomplished on the basis of these efforts.
According to first aspect of the present invention, there is provided a polypeptide which is a Fas ligand.
According to second aspect of the present invention, there is provided a fragment of the Fas ligand polypeptide according to the first aspect of the invention.
According to third aspect of the present invention, there is provided a DNA fragment containing a nucleotide sequence coding for the polypeptide according to the first or second aspect of the invention.
According to fourth aspect of the present invention, there is provided a recombinant DNA molecule including the DNA according to the third aspect of the invention.
According to fifth aspect of the present invention, there is provided a transformant which has been transformed with the DNA fragment according to the third aspect of the invention.
According to sixth aspect of the present invention, there is provided a transformant which has been transformed with the recombinant DNA molecule according to the fourth aspect of the invention.
According to seventh aspect of the present invention, there is provided a process for producing the polypeptide according to the first or second aspect of the invention wherein the transformant according to the fifth or sixth aspect of the invention is used.
According to eighth aspect of the present invention, there is provided a process for purifying the polypeptide of the first or second aspect of the invention.
According to ninth aspect of the present invention, there is provided an antibody capable of recognizing the polypeptide according to the first or second aspect of the invention.
According to tenth aspect of the present invention, there is provided an oligonucleotide or a derivative thereof which includes a nucleotide sequence which is complementary to a part of a Fas ligand gene or a part of mRNA for the Fas ligand.
According to eleventh aspect of the present invention, there is provided a process for screening a Fas ligand-related substance wherein the polypeptide according to the first or second aspect of the invention, or a transformant capable of expressing the polypeptide according to the first or second aspect of the invention is used.
Other objects and advantages of the present invention will become apparent as the description progresses.